Recent studies have suggested that progestins play a role in the etiology of breast cancer; however, the mechanisms by which progestins promote tumor formation/progression have not been defined. Progestin action is mediated by the progesterone receptor (PR), which has been shown to mediate both growth stimulatory and growth inhibitory effects of progestins in breast cancer cell lines. These observations highlight the complexity of PR signaling in breast cancer and the need for further study of the regulation of PR function. Studies of PR target gene expression in breast cancer cells are typically performed with cells grown under serum conditions that arrest them in G1, and our previous studies suggest that PR activity is likely to have unique activities in the various phases of the cell cycle, possibly through differential co-regulator recruitment. Our previous studies have also shown that nuclear translocation of PR is different across the cell cycle, with a greater amount of translocation in S-phase, compared to G2/M. This observation suggests that rapid signaling, which does not require PR binding to DNA, might be highest in G1 or G2/M where residual PR is found in the cytoplasm. Furthermore, our studies suggest that cyclin A/Cdk2/Cdk1 activity plays a role in S-phase gene expression. I will determine whether PR activity differs as a function of cell cycle by 1) using microarrays to identify genes that are differentially expressed across the cell cycle, 2) assessing whether PR-induced rapid signaling is cell cycle-dependent and 3) depleting cyclin A2 using siRNA or by using Cdk1/2 inhibitors to ask whether S-phase genes are preferentially sensitive to cyclin A/Cdk activity. I will also utilize novel, high througput microscopy analyses to 1) perform an immunofluorescence screen to identify novel PR co-regulators, 2) determine whether recruitment of these co-regulators differs as a function of cell cycle, and 3) examine the effects of cell signaling and Cdk-dependent pathways on co-regulator recruitment using MAPK and Cdk inhibitors. Collectively, these studies will contribute to our knowledge of the regulation of PR activity. Understanding how PR is regulated in breast cancer is important because PR mediates breast cancer cell proliferation directly by regulating target gene expression and indirectly through cross-talk with other mitogenic cell signaling pathways. Investigation of the factors necessary for PR activity may eventually unveil potential therapeutic targets for breast cancer. PUBLIC HEALTH RELEVANCE: Progestins, signaling through the progesterone receptor, have been linked to breast tumor formation/progression. Progesterone receptor function is modulated by numerous factors, many of which are not completely understood. Understanding how progesterone receptor activity is regulated may unveil novel therapeutic targets for breast cancer.